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Pharmacokinetics and methaemoglobin reductase activity as determinants of

species susceptibility and non-target risks from sodium nitrite manufactured

feral pig baits

Steven J. LapidgeAD and Charles T. EasonBC

AInvasive Animal Cooperative Research Centre, 48 Oxford Street, Adelaide, SA 5061, Australia.BBio-Protection and Ecology Division, Lincoln University, PO Box 84, Lincoln 7647, Canterbury,

New Zealand.CConnovation Ltd, PO Box 58613, Manukau 2141, New Zealand.DCorresponding author. Email: [email protected]

Abstract. Vertebrate pesticides are used in Australia to manage populations of invasive pest

species, including feral pigs, which threaten native ecosystems, damage crops and spread disease.

Feral pigs are currently poisoned using actives that are considered by some to be inhumane and

more suitable agents are being sought. Pigs are susceptible to methaemoglobin forming compounds,

due to innately low levels of methaemoglobin reductase, and research has identified that sodium

nitrite is a promising humane alternative active. To attain registration, a new vertebrate pesticide

must however be species-specific in its toxicity (rare), or be presented in such a manner to

demonstrate that it is safe for use around non-target species. Furthermore, it should be shown to

represent a low risk of bioaccumulation and secondary poisoning, as is the case with sodium nitrite

which has a half-life of one hour or less in those species tested. Here we present a risk analysis for

the use of sodium nitrite used in manufactured feral pig baits. As pharmacokinetics of sodium

nitrite are similar in different species, and it does not require biotransformation to an active

metabolite, methaemoglobin reductase activity in red blood cells from different species is directly

correlated with differences in acute toxicity. Primary poisoning risks, or susceptibility, have been

calculated for 28 marsupial and nine eutherian mammal, four reptile and two bird species based on

published doses and methaemoglobin reductase activity levels. Those species that have been

previously recorded to sample manufactured feral pig baits, and will be susceptible to the likely bait

dose used, are identified, along with the percentage of a bait that they will need to consume. The

current steps being taken to limited potential non-target poisonings, including an appropriate active

to matrix ratio, are discussed.

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Sodium nitrite in HOG-GONE® risk analysis

INTRODUCTION

Currently, sodium fluoroacetate (1080), warfarin and yellow phosphorus are the toxins used to

poison feral pigs, Sus scrofa, in Australia. However, the use of warfarin and yellow phosphorus has

recently been assessed as inhumane (Cowled and O’Connor 2004; Sharp and Saunders 2004).

Sodium fluoroacetate is the most commonly used toxin, with it being added to various palatable bait

substrates for poisoning pigs, such as grain, pellets, meat and manufactured baits (Hone and Kleba

1984; Twigg et al. 2005; Cowled et al. 2006a). But whilst it appears markedly more humane than

other current pig toxins (Cowled and O’Connor 2004; Sharp and Saunders 2004), it has come under

scrutiny and is not favoured by animal welfare groups (Sherley 2004 and 2007; Cooper et al. 2007).

In 2005 a search was undertaken for other potential chemicals that would be more suitable for feral

pig management, with sodium nitrite proving to be the lead candidate (Cowled et al. 2008a). Proof-

of-concept pen trials showed that it was highly efficacious in euthanasing pigs whether given by

gavage or eaten in PIGOUT® baits (Cowled et al. 2008a). Ironically, it is the same chemical that is

used to preserve pig meat that is also highly toxic to the live animal. Because of the widespread use

of sodium nitrite as a human food preservative there is a considerable amount of data on the

chemistry and toxicology of this compound. Nitrite acts by the humane (Porter and Kuckel 2009),

and Royal Society for the Prevention of Cruelty to Animals (RSPCA) supported (Sherley 2007),

mode of action of methaemoglobinaemia, and so is preferred to 1080 on welfare grounds (Marks et

al. 2004; Cowled et al. 2008a). Another methaemoglobin former, para-aminopropiophenone

(PAPP), is currently being developed for foxes, wild dogs and feral cats in Australia (Marks et al.

2004; Fleming et al. 2006), and stoat, ferrets and feral cats in New Zealand (Fisher et al. 2005;

Fisher and O’Connor 2007; Murphy et al. 2007). Pigs are highly susceptible to the effects of

methemoglobinaemia as they contain uniquely low levels of methemoglobin reductase (Smith and

Beutler 1966; Agar and Harley 1972), the enzyme required to reverse the methemoglobin formation

process. This inherent weakness has previously resulted in numerous reported fatal cases of

domestic pigs being poisoned with nitrite (Robinson 1942; Gwatkin and Plummer 1946; Winks et

al. 1950; London et al. 1967).

As with PAPP, nitrite kills through terminal hypoxia caused by methaemoglobinaemia. High

methaemoglobin levels reduce the oxygen-carrying capacity of the blood and at toxic doses hypoxia

and central nervous system depression precede death. Time to death for feral pigs is 1-2 hours with

few visual symptoms (Cowled et al. 2008a; Lapidge et al. 2009). This is a marked improvement on

conventional vertebrate poisons. A further benefit of nitrite is that it is known to break down readily

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in the environment through biological reduction (e.g. Wanntorp and Swahn 1953; Sofia et al. 2004),

limiting non-target poisoning risks and environmental contamination. A further benefit of the

chemical is that methylene blue will reverse methaemoglobinaemia induced by sodium nitrite, or

PAPP, and is an established antidote for nitrite poisoning in livestock (Wendel 1939; Wright et al.

1999).

Understanding mechanisms of toxicity, pharmacokinetics and receptor level effects are important

for a number of reasons (Eason et al. 1990). These include development of antidotes, risk

assessment, defining the susceptibility of non-target species, defining the risk of secondary

poisoning or bioaccumulation, and also the risk of residues persisting in non-target species,

including wildlife and livestock inadvertently exposed to sub-lethal doses. In the following sections

we firstly examine the basis for species variation in response to xenobiotics, and then review the

pharmacokinetics of sodium nitrite and then receptor level processes as determinants of species

susceptibility. This risk assessment is based to some extent on the research into the sensitivity of

Australian animals to 1080 poison (McIlroy 1981, 1986).

It is planned that formulated sodium nitrite will be delivered using the new HOG-GONE® matrix

(Animal Control Technologies Australia P/L). The concept is based on the successful PIGOUT®

bait matrix which is currently used to deliver 1080 to feral pigs (Cowled et al. 2006a,b), but was

also designed as a potential vaccine carrier (Campbell et al. 2006; Cowled et al. 2008b).

Improvements to the target-specificity of feral pig baiting campaigns have been substantial in

Australia when compared to the use of grain and meat baiting materials (Cowled et al. 2006b).

Preliminary field trial results for the HOG-GONE® matrix, which is harder and less pungent,

indicate that the target specificity has been increased further (S. Lapidge, unpub. data; A. Bengsen,

unpub. data). Notwithstanding, a thorough risk analysis is required for the potential field use of

sodium nitrite if the chemical is to gain registration as a new vertebrate pesticide. This paper

predicts the innate susceptibility to sodium nitrite for 28 marsupial and nine eutherian mammal,

four reptile and two bird species based on published oral doses and methaemoglobin reductase

activity levels. It highlights non-target species potentially at risk for the use of HOG-GONE® baits,

and the methods being employed to minimise such risk. The risks of secondary poisoning, either

through the consumption of sub-lethally dosed or poisoned animals are also discussed.

Primary poisoning

Pharmacokinetics and species variation

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The metabolism and pharmacokinetics of a vertebrate pesticide are often determinant factors in its

ultimate manifestation of toxicity, non-target safety and residue profiles. The metabolic deactivation

of toxins results from the actions of enzymes whose primary function is to protect the body against

the accumulation and undesirable effects of foreign compounds naturally present in food and in the

environment. Metabolism and excretion are protective processes attempting to limit persistence in

the body.

Species differences in sensitivity to an individual chemical are linked to variation in the

pharmacokinetic differences. Savarie et al. (1983) clearly demonstrated this with PAPP, with a

seventy-fold difference occurring between the LD50 of a coyote and a Striped skunk or Golden

eagle. Wood et al. (1991) later detailed that this was due to the metabolic activation of PAPP to a

reactive, and more toxic, intermediate metabolite in susceptible species such as canines. This is also

the case with 1080 and cholecalciferol. However, as is normal with toxic intermediates of other

drugs or pesticides, metabolic processes in the body then go on to detoxify these intermediate

compounds as well. In the case of 1080 and sodium nitrite the parent compound is itself highly

water soluble; hence the normal metabolic processes that render a toxin more water soluble are less

important with regard to their elimination from the body.

Receptor site interactions and species susceptibility

The pharmacokinetics of the direct methaemoglobin (MetHb) former sodium nitrite is similar in

different species as it does not require biotransformation to an active metabolite (Wright et al.

1999). As with the drug amrinone (Eason et al. 1986), there are rapid and similar patterns of

absorption and excretion across a range of different species. Hence, extrapolation of chemical-

receptor interactions can be used with greater confidence to predict the innate risk of sodium nitrite

to non-target species. The receptor site for sodium nitrite poisoning is haemoglobin in the red blood

cell. The mode of action of nitrite is the oxidization of the haem iron in red blood cells from the

ferrous state (Fe++) to the ferric state (Fe+++) to form MetHb. MetHb is incapable of carrying oxygen

and cyanosis results, with death occurring if the dose is high enough (Egyed and Hanji 1987). The

pattern of methaemoglobinaemic response induced when erythrocytes are exposed to sodium nitrite

oxidant challenge will be a balance between MetHb formation and its subsequent reduction back to

haemoglobin by the protective enzyme MetHb reductase.

The activity of the enzyme MetHb reductase varies in different animals, and is known to determine

a species direct sensitivity to a direct methaemoglobin former (Smith and Beutler 1966; Stolk and

Smith 1966; Agar and Harley 1972; Board et al. 1977; Lo and Agar 1986; Whittington et al. 1995;

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Agar et al. 2000; Rockwood et al. 2003). Species differences in MetHb reductase are therefore

critical when evaluating the risk to different species from MetHb formers such as sodium nitrite.

Under normal conditions this enzyme is the only system within the erythrocyte that maintains

haemoglobin in its oxygen-carrying reduced state. Toxicologically, MetHb reductase will therefore

be the rate-limiting enzyme controlling the toxicodynamics of sodium nitrite’s effect on the red

blood cells. In theory, species with lower MetHb reductase activity convert MetHb back to

haemoglobin more slowly than do species with higher activity, and will therefore be more

susceptible to sodium nitrite. Conversely animals with greater MetHb reductase activity will be at

less risk of sodium nitrite induced toxicity.

Correlating methaemoglobin reductase activity, body size, diet and risk

To test this hypothesis we collated all published acute oral sodium nitrite toxicity data and MetHb

reductase activity, as generally determined through nitrite challenge. Data for seven eutherian

mammal species were obtained (Table 1). In those species for which published data exists on both

gavage acute toxicity and on red blood cell MetHb reductase activity there is a strong correlation

between susceptibility and MetHb reductase activity (r=0.894). Simple linear regression analysis

revealed a highly significant (F1,5= 21.7; P = 0.006) relationship, with a line of best fit accounting

for 81% of the variance with the equation y=0.2614x-10.149 (Fig. 1). The equation was

subsequently used to predict the lethal oral dose of sodium nitrite for all Australian species with

published MetHb reductase activity levels. MetHb reductase activity values were obtained from

Agar et al. (2000), except for the platypus and echidna (Whittington et al. 1995). Reptile and bird

values were obtained from Board et al. (1977), and eutherian mammal values from Lo and Agar

(1986) and Rockwood et al. (2003). Predictions were based on the equation:

Estimate minimum lethal gavage dose = (MetHb reductase activity + 10.149) / 0.2614)

and results are presented in Table 2.

Estimated lethal bait dose is based on a correction factor of 3 times, as indicated between the

minimum effective gavage and bait delivered sodium nitrite doses for feral pigs (Cowled et al.

2008a). The correction factor takes into account the destruction of nitrite by stomach acid and

slower absorption when delivered in the physically dense PIGOUT/HOG-GONE® bait matrix. This

correction factor would change if another bait type was used, a smaller volume of matrix was used,

or a high active to matrix ratio was used. It may also differ between species depending on the

digestive physiology of the species.

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The indicative body weights used in Table 2 to estimate lethal bait-delivered sodium nitrite doses

are the average adult body mass of males and females as published in Strahan (2004), as well as

various internet sources. It is acknowledged that juveniles of each species will require smaller doses

of sodium nitrite to those predicted for adults. Species recorded consuming PIGOUT® baits were

reported by Cowled et al. (2006b) or are unpublished data. Early indications are that the HOG-

GONE® bait, which will carry sodium nitrite, is significantly more target-specific with introduced

red foxes (Vuples vulpes)and Australia ravens (Corvus coronoides) the only non-target species

recorded consuming toxic bait during preliminary fieldtrials. Although many of the species listed in

Table 2 are highly unlikely to consume a manufactured feral pig bait, they have been included in

case other bait matrices are considered for the delivery of sodium nitrite in the future, such as grain.

- Insert Table 1 -

- Insert Figure 1 –

- Insert Table 2 -

Proofing model: direct non-target species testing

To examine the validity of the lethal dose predictions in Table 2, and to ascertain the direct risk to

two key potential non-target species that have previously been observed to consume PIGOUT® baits

(Lapidge, pers. obs. and Cowled et al. 2006b), direct toxicity testing on Common brushtail possums

and Tammar wallabies was undertaken by Landcare Research (New Zealand) and Connovation

(New Zealand) respectively. Trials occurred in New Zealand where both species are invasive and

the use of sufficient animals for potentially lethal trials was more likely to be ethically approved.

Both trials were approved by each organisations respective Animal Ethics Committee.

Pen trials were undertaken on 12 individually caged brushtail possums at the Landcare Research

Animal Facilities in Lincoln, New Zealand. Although the trial was to be conducted using non-toxic

and toxic HOG-GONE® baits, the use of the animal protein-containing baits was blocked by the

New Zealand Ministry of Agriculture and Forestry, and as such a substitute but very similar bait

matrix (Connovation Ferafeed 213) was used for the trials. Formulated sodium nitrite was hand-

incorporated into the bait matrix at the same 10% toxin ratio as HOG-GONE® so the results were

directly applicable. Possums were acclimatised to the facility for four weeks prior to the trial

commencing.

On the day prior to toxic testing each possum was offered 125g of non-toxic bait material, of which

they consumed 32±1g on average over 22 hours. The following day each possum was offered four

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25g toxic matrix balls (notably easier to consume than one 200g HOG-GONE® solid bait encased in

a cellulose skin), of which they ate 3.8±0.5g on average over 11.5 hours. The total amount of each

bait type was substancially different, indicating that the toxic bait was less acceptable to possums.

Maximum consumption by any individual possum was 8g of matrix (0.8g active) or 229 mg/kg of

sodium nitrite. Although five of the 12 possums showed visible signs of methaemolobin formation

(blue/cyanotic nose), none of the animals were affected behaviourally in terms of responses to

stimuli, and as predicted no possums received a lethal dose and died. The model predicted a

minimum lethal dose of 393 mg/kg or 1.6g of active (Table 2). All possums were monitored for

seven days following the trial, with the maximum weight changes being -3.3% to +10.5%, with all

possums appearing healthy throughout the observation period (Fisher et al. 2009).

Tammar wallaby trials were undertaken in outdoor pens in a new animal research facility at

Rotorua. Twelve individually-housed wallabies were fed a single 10g ball of Ferafeed (permission

to us HOG-GONE was again denied), instead of their 100g of stock pellets, on three days leading

up to toxic testing. Water and grass was available ad libitum. All wallabies were observed to

consume their 10g ball of Ferafeed within 45 min. Toxic trials consisted of the presentation of 50g

of the 10% nitrite matrix presented in four small balls. Nine of 12 wallabies would not consume any

toxic matrix after sniffing the mixture. Three wallabies that consumed material (body weights 1.9,

3.0 and 4.0 kg) ate 17, 10 and 25g of matrix respectively, resulting in nitrite doses of 894, 335 and

625 mg/kg respectively. All doses were lethal, as predicted in Table 2 (minimum lethal dose 245

mg/kg), with the mean time to first symptoms being 63 min and 157 min to death. Of note is that

one juvenile 3kg female consumed only 1g of sodium nitrite, which is less that the estimated lethal

bait dose of 1.6g predicted in Table 2. This discrepancy is due to the adult tammar wallaby weight

of 6.5kg used to make the prediction (from Strahan 2004). The three wallabies that died all

displayed lethargy, shallow breathing, slight leg spasms, and unconsciousness before death (Shapiro

and Eason 2009).

Additional non-toxic trials were undertaken by the Tasmanian Department of Primary Industries

and Water to assess the attractiveness of the HOG-GONE® baits to Bennett’s wallaby (Macropus

rufogriseus; n=5) and Tasmanian pademelon (Thylogale billardierii; n=17) held at the departments

Launceston facilities. Twelve HOG-GONE® baits were placed in the wallabies 0.8ha enclosure and

monitored using motion sensitive video cameras continually for 5 nights. Although most wallabies

investigated the baits at least once, they generally recoiled abruptly once smelling the bait material

(Fish and Statham 2009). No wallabies showed any inclination to consume the baits.

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Although low sample sizes for possums (n=5) and tammar wallabies (n=3), results from toxic trials

supported the lethal dose predictions made in Table 2, and provide some validity to the model

approach presented. Results from the overall trials do however clearly indicate that marsupials are

generally repelled by the HOG-GONE® bait matrix itself or the smell of formulated sodium nitrite.

Despite formulation, the nitrite smell is still present in the toxic matrix and the chemical is

extremely salty to taste. This is likely to be accentuated in the final product when the two are

combined, although additional masking formulation research is still ongoing. Notwithstanding, the

primary poisoning risks posed by a toxic HOG-GONE® baiting campaign are likely to be

acceptable, particularly considering that in a recent HOG-GONE® field trial on Kangaroo Island,

South Australia, no free-living tammar wallabies were recorded consuming ground-laid and

unprotected HOG-GONE® baits, despite the species being locally abundant (Lapidge, unpub. data).

Secondary poisoning

Pharmacokinetics of sodium nitrite

There is no evidence that sodium nitrite exerts selective toxicity based on classical species variation

in the metabolism or pharmacokinetics of this compound in mammals. Analysis of the publications

on the fate of sodium nitrite in animals and humans therefore enables comparisons across different

species. All these publications indicate that sodium nitrite is rapidly eliminated by different animals

and humans. Since sodium nitrite does not require biotransformation to be active there is a close

correlation between the concentration of sodium nitrite in the blood and methaemaglobin induction.

One of the more detailed recent publications in this field by Kohn et al. (2002) examines time and

the dose-dependent relationship between exposure to nitrite and induction of methemoglobinemia in

rats. The authors report on the toxicodynamic processes as absorption of nitrite following oral

ingestion, elimination from the plasma, partitioning between plasma and erythrocytes, binding of

nitrite to haemoglobin and methaemoglobin, and the free radical chain reaction for haemoglobin

oxidation. Peak plasma levels of nitrite were achieved in both sexes of rats approximately 30

minutes after oral exposure, and peak methaemoglobin levels were achieved after 100 minutes. The

t(1/2) for recovery from methemoglobinaemia was 60 to 120 minutes depending on dose and route

of administration.

We have reanalysed the raw data from Kohn et al. (2002) to elucidate the time course of plasma

nitrite in rats at different doses and this is summarised in Table 3. The plasma elimination t(1/2) for

sodium nitrite in rats ranges from 42.0 to 62.5 minutes after oral dosing. These values are similar to

those quoted in humans (Dejam et al. 2007), who showed a plasma elimination half-life of 42

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minutes. They also correlate closely to plasma elimination t(1/2) values of 29.0, 30.0 and 34.0

minutes in sheep, dog and ponies reported by Schneider and Yeary (1975).

- Insert Table 3 -

In an unpublished report (MRI 2004) confirmation that sodium nitrite is rapidly eliminated from the

blood is provided in both rats and mice. In rats orally dosed with 80 mg/kg peak plasma

concentrations were achieved at 30 mins after dosing and these decreased to below the limit of

detection after 8 hours. In mice peak plasma concentrations occurred after 10 mins and sodium

nitrite was undetectable in the blood after 4 hours. Earlier publications on the biotransformation of

sodium nitrate indicate that conversion to nitrite is important for toxicity, however nitrite is very

quickly converted and may not be readily detected, therefore methaemoglobin formation was often

used as an indicator of nitrite formation (Ward et al. 1986). The rapid elimination of nitrite

predicted by Ward et al. (1986) has been confirmed by Kohn et al. (2002) and other research

groups. Nitrite is reportedly broken down to hydroxylamine, ammonia and urea (Mascher and

Marth 1993). Kovacs et al. (1960) reported that the concurrent administration of sodium

bicarbonate was found to increase the lethality of sodium nitrite, probably due to the creation of low

acid conditions in the stomach, reducing breakdown to ammonia and aiding absorption.

The pharmacokinetic data on sodium nitrite in such diverse species as mice, rats, sheep, dog, ponies

and humans, coupled with information on the toxicodynamics of sodium nitrite, suggests similar

Cmax, t(1/2), and rapid clearance would occur in other animal species and we could expect

elimination of sodium nitrite following sub lethal exposure within 12 hours.

To help distinguish between different vertebrate pesticides and add some clarity to risk assessment,

Eason et al. (2008) has recently classified compounds used for animal pest control into four groups,

based on their persistence in sub-lethally exposed animals. The criteria for the four groups, and the

allocation of different compounds to these groupings, are described below and in Table 4.

Group 1 – Sub-lethal doses of these poisons are likely to be substantially excreted within 24 hours (e.g.,

cyanide, zinc phosphide, PAPP and 1080). In the case of 1080, complete excretion of all residues may

take up to 4-7 days. On the basis of what we can identify in the literature regarding sodium nitrite we

believe it appropriate to include it in Group 1 and distinguish it from the more persistent compounds in

Groups 2-4.

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Group 2 – Residues resulting from sub-lethal doses of these poisons are likely to be substantially

cleared from the body within 2 to 4 weeks (e.g., pindone and diphacinone).

Group 3 – Residues resulting from sub-lethal doses of these poisons are likely to be cleared from the

body within 2 to 4 months (e.g., cholecalciferol and coumatetralyl).

Group 4 – Residues resulting from sub-lethal doses of these poisons may not ever be completely

cleared from the body (e.g., bromodiolone, brodifacoum, difenacoum, and flocoumafen).

- Insert Table 4 -

Risk to non-target species from vertebrate pesticides can be exacerbated if they bioaccumulate in

the food chain. Pharmacokinetic analyses in different animals and humans have shown that unlike

some common rodenticides e.g. brodifacoum, sodium nitrite is unlikely to cause secondary

poisoning due to bioaccumulation. Furthermore it is unlikely to cause any significant food web

contamination when used repeatedly as a vertebrate pesticide because it will be rapidly excreted.

DISCUSSION

This paper has demonstrated that the toxicity of sodium nitrite is directly related to the activity of

methaemoblobin reductase in each species. This strong relationship has meant that a risk analysis

for 43 species of mammals, birds and reptiles residing in Australia has been able to be undertaken

without the need for extensive lethal trials, as often previously occurred in the past (McIlroy 1981).

Although not as extensive as the risk assessment research documented by McIlroy (1986) for 1080,

c. 171 species, the analysis provides a clear indication of those species that are likely to be at risk.

Results from this analysis indicate that sodium nitrite is highly toxic to most species. Although feral

pigs are one of the more susceptible species on a mg/kg basis (Table 1; Cowled et al. 2008a), they

are also a large animal compared to most potential non-target species, and therefore the amount of

chemical required to humanely euthanase an adult feral pig will always mean that non-target species

will be at risk unless the active can be delivered in a species specific manner. The HOG-GONE®

bait has been demonstrated to almost achieve this, with the exception of foxes and ravens.

Obviously the same risk analysis will need to be undertaken for any other manufactured baits, or if

a concentrate version of the chemical was made for presentation in grain or meat baits.

This risk assessment has involved all marsupial and eutherian mammal, reptile and bird species

occurring in Australia for which methaemaglobin reductase activity has been published. Besides

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foxes and ravens, the risk analysis covers most species that have previously been recorded to take

manufactured feral pig baits. Obviously many of the species listed in Table 2 are highly unlikely to

consume manufactured feral pig baits, but have been included should predictions ever be required.

Potential exists for other species to be included if data on direct susceptibility or methaemaglobin

reductase activity is found. Furthermore, non-lethal assessment of methaemaglobin reductase

activity can be easily undertaken to assess other species innate sensitivity (Power et al. 2007). Such

a process will need to be used if sodium nitrite is to be registered in other countries where less

information is available on methaemaglobin reductase activity in potential non-target species.

Based on their previous consumption of PIGOUT® baits the non-target risk posed by a HOG-

GONE® baiting campaign are principally to brush-tailed possums (9% of a bait), northern brown

bandicoots (6%), swamp (17%) and tammar (8%) wallabies, foxes (unknown) and wild dogs (18%).

This is however likely an over-estimation, as recent HOG-GONE® pen and field trials have

indicated that wallabies (swamp, tammar, Bennett’s and Tasmanian pademelon) and wild dogs are

less inclined to consume the new HOG-GONE® bait matrix. Furthermore, given the high intake of

non-toxic HOG-GONE® baits in field trials conducted thus far, scope exists to reduce the individual

bait loading to 10g of formulated nitrite or a 5% ratio. This would effectively double the predicted

bait consumption percentages required in Table 1, and provide a further safeguard. This is because

MetHb needs to be induced rapidly to case death. Target species such as pigs devour their food

quickly which aids induction of lethal MtHb levels, whereas small mammals or birds that nibble at

baits would be more likely to experience a transient sub-lethal MtHb rise. For example, an adult

possum that requires a 1.9g nitrite dose will need to eat 40g of toxic HOG-GONE® matrix in one

short sitting to receive a lethal dose. Results from the Landcare Research trial indicated that only

the largest of the possums would generally do this with non-toxic matrix, with 59g being the highest

consumption over a 22hr period. Furthermore, an animal must consume the dose before it starts

feeling lethargic (the first symptom of nitrite poisoning; Cowled et al. 2009), after which animals

generally stop eating. The slow consumption of baits is unlikely to be lethal in most species as

methaemoglobin reductase is able to keep pace with the conversion of methaemoglobin back to

oxyhaemoglobin. Notwithstanding, methods being used to limit the risk include:

1. the 200g size and formulation of the HOG-GONE® bait itself, whereby most possums and

wallabies are adverse to consuming the bait, particularly when it contains sodium nitrite;

2. adult feral pigs are known to consume multiple HOG-GONE baits, up to 15 in one sitting,

and therefore the lethal dose for particularly large (100kg+) boars can be spread over

multiple baits; and

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3. where necessary, physical exclusion of non-target species is possible, either using large

rocks, over-turned buckets pegged into the ground (as occurs in Namadgi National Park), or

through a bait hopper, such as the Boar Buffet® (Lapidge et al. 2009), that physically

excludes non-target species.

When used as a package there is little primary poisoning risk from the use of HOG-GONE® to non-

target wildlife species or domestic stock. Any baiting campaign will however hold some risk, as

individual behaviour of animals can not always be predicted.

Phamacokinetic analyses of sodium nitrite has demonstrated that it is rapidly eliminated from the

body. Lack of persistence is an important feature in vertebrate pesticide risk assessment. Risk to

non-target species from vertebrate pesticides can be exacerbated if they bioaccumulate in the food

chain. Pharmacokinetic analyses in different animals and humans have shown that, unlike some

common rodenticides (e.g. brodifacoum), sodium nitrite is unlikely to cause secondary poisoning

due to bioaccumulation. The plasma elimination half-life in a wide range of animals, including

mice, rats, dog, sheep and ponies and in humans is approximately 1 hour or less, and residues will

not persist in sub-lethally exposed animals for >12 hours. Given this remarkable consistency in the

pharmacokinetics of sodium nitrite, and the fact that biotransformation is not necessary for sodium

nitrite to be active or toxic, it is possible to elucidate species differences in terms of receptor

differences.

In this paper we have reviewed risk to non-target species linked to pharmacokinetics and also

MetHb reductase activity, animal size and behaviour to determine non-target risk when sodium

nitrite is potentially used for feral pig control in manufactured baits. One aspect we have not

completely covered is the persistence of nitrite in poisoned pigs’ carcasses and the rate of decay of

nitrite. This will require further study to ensure persistence of nitrite is not a feature of its use. This

is a recognised hazard associated with 1080 and clarification of the risk to non-target species form

poisoned carcasses will be needed. Residue data will be ascertained from field poisoned feral pigs

during large-scale HOG-GONE® field efficacy trials and will be published at a later date. As the

reaction of nitrite with deoxyhemoglobin results in the production of nitric oxide and

methemoglobin, most nitrite is converted during the toxicosis, and the chemical has a short half-life,

it is predicted that residues will be negligible.

Sodium nitrite has the potential to become a new, humane (Porter and Kuckel, 2009) vertebrate

pesticide for the management of feral pigs and potentially other species. However, the active is

generally toxic to most species, and will therefore require presentation in species-tailored baits.

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Bengsen et al. (2008) details a method for achieving this. As outlined above, given an appropriate

presentation the active can be delivered in a highly species-tailored fashion that posses few risks for

potential non-target species. The short half-life of the compound and rapid elimination from the

body further reduce this risk. Similarly, nitrite is known to break down readily in the environment

through biological reduction (e.g. Wanntorp and Swahn 1953; Sofia et al. 2004), and is therefore

unlikely to cause persistent environmental residues.

ACKNOWLEDGEMENTS

The preparation of this risk analysis, including the contract testing of possums (Landcare Research,

New Zealand) and tammar wallabies (Connovation, New Zealand), was funded by the Federal

Department of the Environment, Water, Heritage and the Arts. The authors thank Michelle Smith

and the team at Animal Control Technologies Australia P/L, Penny Fisher and team, Lee Shapiro,

Duncan MacMorran, Mick Statham and Rebecca Fish for being directly involved in pen trials.

Associate Prof Chris Frampton, University of Otago, is thanked for his advise on pharmacokinetics

analyses on the Kohn et al. (2002) paper, and Jason Wishart, IA-CRC, is thanked for extracting the

data from this paper which greatly assisted in our understanding of sodium nitrite pharmacokinetics.

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Table 1. Published oral sodium nitrite LD50 doses/minimum lethal doses and NADH

metheamoglobin reductase activities for eutherian mammals.

Species Scientific name Lethal gavage

dose (mg/kg)

NADH methaemoglobin

reductase (IU/g Hb)A

Lethal dose reference

Pig Sus scrofa 90 12Winks et al. 1950; Cowled

et al. 2008

Human Homo sapien 94 15 Boink and Speijers 2001

Rat Rattus norvegicus 130 10 Druckery et al. 1963

Mouse Mus musculus 215 53 Rieman 1950

Dog Canis lupus familiaris 60B 8.5 Berlin 1970

Sheep Ovis aries 50 10 Rhadostis et al. 2000

Cattle Bos taurus 67 5 Bartik and Pisac 1981A NADH methaemoglobin reductase activity values were for adults from Lo and Agar (1986) and Rockwood et al.

(2003).B Sub-cutaneous LD50 of 50-70 mg/kg used as no oral dose could be found (Berlin 1970)

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Table 2. Estimated lethal bait-delivered sodium nitrite doses of marsupials, reptiles birds and eutherian mammals with known

methaemoglobin reductase activities. Species that have consumed manufactured feral pig baits and are of concern are highlighted.

Species Scientific nameNADH-MR (IU/g Hb)

Est. lethal gavage dose

(mg/kg)

Est. lethal bait dose (mg/kg)

Body size (kg)

Est. lethal SN dose

(g)Consumed PIGOUT

% of 200g 10% active SN

baitMarsupialsAllied rock-wallaby Petrogale assimilis 13.2 85 255 4.5 1.1 No N/ABarrow Island wallaroo Macropus robustus isabellinus 15.1 90 270 35.8 9.7 No N/ABilby Macrotis lagotis 13.6 86 258 1.8 0.5 No N/ABlack-striped wallaby Macropus dorsali 13.8 86 259 11.2 2.9 No N/ABridled nailtail wallaby Onychogalea fraenata 19.8 102 307 6.5 2.0 No N/ABurrowing bettong Bettongia lesueur 36.7 147 441 1.5 0.7 No N/ACommon brushtail possum Trichosurus vulpecula 30.6 131 393 4 1.6 SA,ACT,NSW 16Common wombat Vombatus ursinus 16.0 92 277 26 7.2 No N/AEastern grey kangaroo Macropus giganteus 17.6 96 289 49 14.2 No N/AEastern wallaroo Macropus robustus robustus 18.2 98 294 35.8 10.5 No N/AEuro Macropus robustus erubescens 14.4 88 264 35 9.2 ACT 92Koala Phascolarctos cinerus 24.6 115 345 10 3.5 No N/AMountain pygmy possum Burramys parvas 45.4 170 510 0.04 0.0 No N/ANorthern brown bandicoot Isoodon macrourus 52.0 188 563 1.6 0.9 Qld 9Northern hairy-nosed wombat Lasiorhinus krefftii 32.2 135 405 31 12.6 No N/APlatypus Ornithorhynchus anatinus 16.8 94 283 1.5 0.4 No N/AProserpine rock-wallaby Petrogale persephone 12.7 84 251 6.2 1.6 No N/ARed kangaroo Macropus rufus 11.0 79 237 46 10.9 No N/ARed-legged pademelon Thylogale stigmatica 26.9 121 363 4.6 1.7 No N/ARufous bettong Aepyprymnus rufescens 13.2 85 255 3 0.8 No N/ARufous hare-wallaby Lagorchestes hirsutus 7.4 69 208 1.3 0.3 No N/AShort-beaked echidna Tachyglossus aculeatus 64.0 219 658 4.5 3.0 No N/ASouthern hairy-nosed wombat Lasiorhinus latifrons 15.0 90 269 26 7.0 No N/ASpectacled hare-wallaby Lagorchestes conspicillatus 24.1 114 341 3 1.0 No N/ASugar glider Petaurus breviceps 42.0 161 483 0.12 0.1 No N/A

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585


Swamp wallaby Wallabia bicolor 9.9 76 228 15 3.4 NSW 34Tammar wallaby Macropus eugenii 12.0 82 245 6.5 1.6 SA 16Whiptail wallaby Marcropus parryi 15.8 92 275 13.5 3.7 No N/AReptileRed-bellied Black Snake Pseudechis porphyriacus 10.0 76 229 0.5 0.1 No N/AWestern Brown snake Pseudonaja nuchalis 12.3 82 247 0.4 0.1 No N/AJohnston's crocodile Crocodylus johnstoni 4.5 62 185 12 2.2 No N/ASaltwater crocodile Crocodylus porosus 5.1 63 190 700 133.1 No N/ABirdDomestic chicken Gallus domesticus 1.0 53 158 2.4 0.4 No N/ADomestic pigeon Columba livia 2.3 56 168 0.4 0.1 No N/AEutheriansCattle Bos taurus 67 201 600 120.6 Qld >12 baitsDog Canis lupus familiaris 60 180 20 3.6 ACT,Qld 36Goat Capra hircus 10 76 229 34 7.8 SA 78Guninea pig Cavia porcellus 5 63 189 0.9 0.2 SA 2Horse Equus caballus 11 79 237 450 106.7 SA >10 baitsMouse Mus musculus 215 645 0.03 0.02 KI >1Rabbit Oryctolagus cuniculus 250 750 2 1.5 No 15Rat Rattus norvegicus 104 312 0.1 0.03 KI >1Sheep Ovis aries 50 150 60 9.0 Once 90

A NADH marsupial methaemoglobin reductase concentrations were from Agar et al. (2000), except for the Platypus and Echidna (Whittington et al. 1995). Reptile and bird values from Board et al. (1977). Eutherian mammals from Lo and Agar (1986) and Rockwood et al. (2003).B Estimated lethal bait dose is based on a correction factor of 3 times, as indicated between the minimum effective gavage and bait delivered doses for feral pigs (Cowled et al. 2008a). This correction factor may change between species depending on the presentation of sodium nitrite and the digestive physiology of the species.C Body weights are the average adult body mass of males and females as published in Strahan (2004) as well as various internet sources.D Species recorded consuming PIGOUT® baits were reported by Cowled et al. (2006b) or are unpublished data. Early indications are that the HOG-GONE® bait, which will carry sodium nitrite, is significantly more target-specific with foxes, possums and ravens the only non-target species recorded consuming bait.

20

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595

40


Table 3. Plasma elimination t(1/2) in minutes with lower and upper 95% confidence limits for

sodium nitrite in rats (calculated by Frampton from data in Kohn et al. 2002).

Dose (mg/kg) Route t(1/2) male (min) t(1/2) female (min)

20 i.v 19.8 (16.9-23.8) 28.3 (24.4-33.8)

40 oral 42.1 (37.6-47.7) 55.5 (47.9-65.8)

80 oral 42.0 (33.9-55.0) 62.5 (58.8-66.7)

21


Table 4. Summary of classification of vertebrate pesticides based on comparative

pharmacokinetics and expectation for persistence of residues in sub-lethally exposed target or

non-target species (adapted from Eason et al. 2008 to include sodium nitrite).

Group Compound Half-life values Likely persistence of residues

after sub-lethal exposure

1 Sodium nitrite < 60 min 12 hours

Cyanide + 12 to 24 hours

Zinc phosphide + 12 to 24 hours

Para-aminopropiophenone + 4 days

1080 < 11 hours 7 days

2 Pindone 2.1 days 4 weeks

Diphacinone 3 days 6 weeks

3 Cholecalciferol 10-68 days 3 months

Coumatetralyl 50-70days 4 months

4 Brodifacoum 130 days 24 months or longer

Bromodiolone 170 days 24 months or longer

Flocoumafen 220 days 24 months or longer

+ No published value found, but likely to be < 12 hours.

22

600

605


Figure 1. General linear regression between published sodium nitrite lethal gavage doses and

NADH metheamoglobin reductase activities for eutherian mammals.

y = 0.2614x - 10.149R 2 = 0.8126

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200 220 240 260

Sodium nitrite lethal gavage dose (mg/kg-1)

NADH

met

Hb re

duct

aste

(IU/

g Hb

)

CowDog

Sheep Human

Pig

Rat

Mouse

23

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pharmacokinetics and methaemoglobin reductase activity as ...€¦ · web viewmetabolism and...

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